Yeast based expression systems such as Pichia pastoris are commonly used to express recombinant proteins Cregg, J. M. et al., in Dev. Ind. Microbiol. 29:33-41; 1988. The P. pastoris expression system uses the methanol-induced alcohol oxidase (AOX1) promoter, which controls the gene that codes for the expression of alcohol oxidase, the enzyme which catalyzes the first step in the metabolism of methanol Cregg J M. et al. in Bio/Technology 11: 905-910; 1993. P. pastoris has potential for high expression levels, efficient secretion of extracellular protein, post-translational modifications such as glycosylation, and growth to high cell densities on minimal medium in bioreactor cultures.
Fed batch fermentation process using Pichia pastoris is described in “Pichia fermentation Process Guidelines” of Invitrogen Co. (San Diego, Calif.), hereafter referred to as control. For production of recombinant proteins transformed Pichia pastoris is grown to a desired high cell density biomass on glycerol as carbon source. Production phase is initiated by feeding of methanol which serves as inducer and sole carbon source to the culture. During biomass generation and production phase ammonia is used to control pH which serves as nitrogen source.
Albeit the various advantages conferred by Yeast Expression Systems, there still exists need for optimizing the nutritionally influenced physico-chemical environment for efficient and maximal recombinant protein production in bioreactors. Achieving high specific productivity is highly desirable. It can be obtained by optimization of initial media composition, methanol feeding strategy and process physicochemical parameters. There are reports wherein ammonium sulfate, ammonium phosphate, di ammonium phosphate, potassium nitrate, urea, corn steep liquor, soya bean meal, cotton seed meal, cane and beet molasses, peptones, meal hydrolysates etc have been used as nitrogen source for bacterial, yeast and fungal growth. Use of different carbon and nitrogen sources for ‘growth’ of a microbe is a prior art.
However, the optimum combination of specifically defined carbon and nitrogen sources for efficient production of recombinant proteins, peptides and enzymes has not been disclosed in prior art literature. For example, WO/2007/005646 discloses ethanol production per se, by cultivating recombinant yeasts on complex growth medium containing complex carbohydrates as well as a variety of cheaper nitrogen sources like corn steep liquor, corn extract, yeast autolysate and urea. Further, this process does not utilize the methanol inducible machinery for growth or production unlike the process developed in the present invention for production of recombinant proteins. Similarly, a U.S. Pat. No. 4,288,554 describes a continuous fermentation process for merely growth of a non GMO Candida species using urea in combination with other sources of nitrogen and basal salt medium. There is no suggestion or teaching whatsoever where urea can be used during fermentation process (batch, fed batch, continuous) using methanol inducible GMO Pichia pastoris for efficient production of recombinant proteins and peptides like human insulin and its analogues or enzymes like lipase.
It has been surprisingly found that the use of defined fermentation medium characterized by controlled addition of certain rich and readily soluble nitrogenous sources such as urea further in optimized concentrations with respect to the residual concentrations of urea as well as residual concentrations of ammonia generated from urea hydrolysis enables higher product, productivity and thus lesser production time. Production of recombinant proteins using E coli has been known for years and the expression system is clearly studied and understood. E coli based expression system has been widely used for the production of molecules like GCSF, HGH, Streptokinase and many other similar biological products. For production of recombinant proteins transformed E. coli is grown to a desired high cell density biomass on dextrose as carbon source. Production phase is initiated by induction using a required inducer and then the culture is just maintained with minimal feeding of nutrients till the end of fermentation.
Fungal cultures had been used since ages for the production of valuable bio-entities like enzymes and other commodity molecules. Fungal cultures like Rhizopus, Aspergillus, Penicillium, etc have been used in classical fermentation for the production of wide variety of enzymes like lipases, amylases, dextranases, etc which are used in food, textile, leather and other such industries. Actinomycetes, known as work-horses for antibiotics production, have being used extensively for the production of several secondary metabolites beneficial for human kind. One of the key properties of fungi and actinomycetes is their property for “bioconversion”, like hydroxylation, esterification etc. The major advantage is that the bioconversions are target specific and products with relatively high purity can be obtained. A classical example is the conversion of compactin to pravastatin.
Methodological improvements known in art include measures relating to fermentation technology, such as stirring or supplying with oxygen, or modification relating to composition of the nutrient media such as the modifying sugar concentrations during the fermentation, down-stream processing changes, or changes related to the intrinsic properties of the microorganism itself etc
It has been surprisingly found that the use of fermentation medium characterized by controlled addition of certain rich and readily soluble nitrogenous sources such as urea enables higher productivity and/or higher rate of bioconversion and thus lesser production time.